Aggression after intermittent ethanol intoxication in mice: Sex differences and modulation via medial amygdala relaxin-3/RXFP3 signaling

Alcohol consumption is strongly associated with aggression and violence in humans, yet the underlying neurobiological mechanisms within key aggression circuits remain poorly understood. In this preclinical study, we examined the effects of intermittent alcohol intoxication on dominance and aggressive behaviors in mice, focusing on sex differences and the potential involvement of the nucleus incertus relaxin-3/relaxin-family peptide receptor 3 (RXFP3) signaling pathway. Using an intermittent ethanol-intoxication protocol, we observed that male mice displayed a transient increase in dominance and aggressive behaviors during acute abstinence, as measured by the tube-dominance and resident-intruder tests, whereas female mice displayed heightened defensive responses. Distinct patterns of neural activation across brain regions, reflected by c-Fos protein expression, were associated with aggression in males, including decreased expression in the medial amygdala (MeA) and increased expression in the ventromedial hypothalamus (VMH), consistent with an established MeA-VMH based aggression circuit. Additionally, the levels of relaxin-3 immunoreactivity in MeA nerve fibers increased in parallel with behavioral recovery, suggesting a modulatory role of relaxin-3/RXFP3 signaling. To test this hypothesis, we bilaterally injected an adeno-associated viral (AAV) vector expressing the selective RXFP3 agonist, R3/I5, into the MeA of male mice. Notably, this chronic localized R3/I5 treatment significantly reduced dominance and aggressive behaviors both before and after alcohol intoxication. Together, these data demonstrate that relaxin-3/RXFP3 signaling in the MeA counteracts alcohol-related aggression in male mice, pointing to this pathway as a potential target for treating impulsive violence associated with alcohol intoxication in humans.